Mass Fragmentographic Analysis of Steroids, Catecholamines, and Amino Acids in Biological Materials B. F. Maume, P. Bournot, J. C. Lhuguenot, and C. Baron Laboratory of General Biochemistry, Faculty of Sciences, 27000 Dijon, France
F. Barbier, G. Maume, M. Prost and P. Padieu Laboratory of Medical Biochemistry, Faculty of Medicine, 27033 Dijon, France
Mass fragmentographic methods are described for quantitative evaluation of some biological metabolites present under physiological conditions in blood or tissue. The use as internal standards of perdeuterated-TMS ethers of studied substances themselves is proposed and discussed for quantitative analysis; a nonbiological isomer of the compound submitted to the analysis is also used. Examples are given for the analysis of rare amino acids in heart cell culture, of endogeneous corticosteroids in rat adrenals and blood, of catecholamines in rat adrenals, of estrogens in nonpregnant woman blood, and of endogeneous metabolites of corticosterone in the rat liver. These metabolites are analyzed at the nanogram range. Preliminary works in some of these fields made with a coupled glass-capillary column are also described.
Progress made in the culture of normal isolated cells from heart (1) and recently from liver (2, 3) and from adrenals offers an excellent model system for the investigation of some biochemical mechanisms such as those of protein synthesis, of hormonogenesis, and of action of sexual steroids on the catabolic function of the liver. But because of the limited size of these biological systems, the amounts of metabolites which can be used as chemical markers of the cell differentiation are a t a very low concentration. For this reason, accurate and sensitive methods of assay which are separative enough to distinguish between several similar chemical structures encountered, for instance, in a same metabolic pathway, are needed. This prompted us to develop methods of analysis of amino acids, steroids, and catecholamines a t the nanogram and subnanogram level from small biological samples by gas-liquid chromatography (GLC), gas-liquid chromatography-mass spectrometry (GLC-MS) and mass fragmentography (MF). GLC methods are now widely used for the analysis of several groups of biological substances because of their high separating power. They allow the separation from a biological sample of a large range of compounds, usually of the same class or linked by the same metabolic pathway, leading to a multicomponent analysis (4). The use of a combined gas chromatograph-mass spectrometer gives a high specificity to the analysis by providing a physicochemical identification of the prepurified GLC effluents
(1) I . Hararyand B. Farley. Science, 131, 1674 (1960). ( 2 ) P. Padieu, C. Lallemant, F. Barbier. and M . Chessebeuf. at the 7th Congress of the Federation of European Biochemical Society, Varna, Bulgaria, 1971. (3) P. Padieu, F. Barbier, M. Chessebeuf, D. Cordier. M. Gerique, C. Lallemant, and A. Olsson, at the First International Conference on Cell Differentiation, Nice, France, 1971. (4) E. C. Horning and M. G. Horning, J. Chromatogr. Sci., 9, 129 (1971).
(5). Furthermore, small metabolites a t low concentration as they are encountered in blood or in small samples of tissue are amenable to evaluation by the gas phase method; it is possible, indeed, to bring the sensitivity of the detection up to this level by using an electron capture detector (ECD) or, better, the mass spectrometer itself as a selective detector of the GLC effluent. In this last method, one (single ion detection SID), two, or more (multiple ion detection MID) fragment ions are monitored by the mass spectrometer during the GLC elution. This allows specific, sensitive, and quantitative detection of the substances where these ions are present. The MID method was first introduced by Sweeley et al. in 1966 (6) and was developed for drug metabolite analysis in blood by Hammar et al. who called it mass fragmentography (7). In the field of steroids, SID has been essentially developed by Brooks et al. for the analysis of steroidal drugs (8, 9) and 11-desoxycortisol as a model compound (10). In the field of catecholamines, Koslow et al. (11) have described a mass fragmentographic assay of norepinephrine and dopamine in the brain. Detection of steroids at low concentration has also been reported by using a GLC-MSComputer system (12). We have already done preliminary reports of GLC, GLC-MS, and M F (13, 14). In the same sense a recent report has been done on the use of MF in biological research (15). This paper describes mass fragmentography with multiple ion detection of rare amino acids of actomyosin from cultured heart cells, of endogeneous steroid and catecholamine hormones from pieces of adrenal tissue, of steroids in blood, and of endogeneous corticosterone metabolites from rat liver.
R. Ryhage, Anal. Chem., 36,759 (1964). C. C. Sweeley, W . H. Elliott, I . Fries, and R . Ryhage, Anal. Chem., 38, 1549 (1966). C. G. Hammar, B. Holmstedt. and R . Ryhage. Anal. Eiochem., 25, 532 (1968). C. J. W. Brooks, A. R. Thawley, P. Rocher. B. S. Middleditch. G. M. Anthony, and N. G. Stillwell, J. Chromatogr. Sci., 9, 35 (1971). C. J. W . Brooks and B. S. Middleditch, Clin. Chim. Acta, 34, 145 (1971). T. A. Baillie, C. J. W. Brooks, and B. S. Middleditch, Anal. Chem., 44, 30 (1972), S. H. Koslow, F. Cattabeni, and E. Costa, Science. 176, 177 (1972). R. Reimendal and J. Sjovall, Anal. Chem., 44, 21 (1972). B. F. Maume,,G. Maume, M. Prost, P. Bournot, J.-C. Lhuguenot, J. Durand, and P. Padieu, in "Proceedings of the Second Congress on Automation and Prospective Biology," Pont-a-Mousson. 1972; Expansion Scientifique Franpaise, Paris, 1973, in press. P. Padieu. F. Barbier, R. J. Begue. P. Bournot, J. Desgres, J . Durand, G. Maume, J. C. Lhuguenot, M. Prost, and 8.F. Maume, in "Proceedings of the Second Congress on Automation and Prospective Biology." Pont-a-Mousson, 1972, Expansion Scientifique Francaise. Paris, 1973, in press. R. E. Gordon and A. Frigerio, J. Chromatogr., 73, 401 (1972) A N A L Y T I C A L C H E M I S T R Y , VOL. 4 5 ,
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EXPERIMENTAL Gas-Liquid Chromatography. Becker Model 420 dual-column instruments equipped with flame ionization detectors and with Sefram model Servotrace recorders were employed for steroid analysis. The columns were 12-ft X 3-mm i.d. silanized glass tubes. The column packings were 1% SE-30 Ultraphase (Pierce Chemical Co.), 1% OV-17 (Supelco Inc.), and 1% Dexsil-300 GC (Analabs Inc.) on 100-120 mesh size-graded, acid-washed and silanized Gas Chrom P (Supelco Inc.). All packings were prepared according to the procedure developed by Horning et a / . (16) Separations were carried out by temperature programming a t a rate of 1.2 "C/min, from 180 "C. The number of theoretical plates was in the range of 5000-9000, the flow rate of nitrogen in the range of 30-40 ml/min. The injector block temperature was 260 "C and the detector bath 280 "C. A Packard Model 7400 dual-column instrument equipped with electron capture detectors and with Texas Instruments Model Servoriter recorders was employed for catecholamine analysis. The columns were 12-ft X 3-mm i.d. silanized glass coiled tubes. The column packings were the same as above. A 5-V direct current was applied to the 63Ni (15 pCi) detector. Separations were carried out by keeping the temperature constant a t 190 "C. The injector block temperature was 250 "C, the detector bath 260 "C, and the pressure of the argon-methane (90: 10) carrier was 22 psi. Capillary Columns. The 20-m X 0.25-mm i.d. glass capillary column coated with SE-30 has been prepared according to the procedure of Rutten and Luyten (17). The number of theoretical plates was about 30,000 for n-octocosane. An all-glass solid injector was used (18) and the flame ionization was employed for detection. Estrogen and corticosteroid separations were obtained by temperature programming from 210 "C a t 1 "C/min and from 235 "C a t 0.8 "C/min, respectively. Gas-Liquid Chromatography-Mass Spectrometry. A LKB Model 9,000, GLC-MS instrument was employed. The chromatographic columns were 3- or 4-m X 3-mm silanized glass coiled tubes coated with 1% SE-30 or 1% OV-17 liquid phases on 100120 mesh size-graded, acid-washed and silanized Gas Chrom P . The flash heater temperature was 260 "C, the molecular separator 270 "C, and the ionization source 290 "C. The flow rate of carrier gas (helium) was 30 ml/min. The accelerating voltage was 3.5 kV and the trap current 60 FA. The mass spectra were recorded a t electron ionizing energies of 70, 28, and 20 eV. An accelerating voltage alternator device was used for multiple ion detection. It allowed detection of three fragment ions up to 20% higher in mass than the lower mass. The energy of the electron beam was adjusted a t 20 or 28 eV. The procedure for the coupling of the capillary column to the mass spectrometer has been recently described (19). Reference Substances a n d Derivatives. Steroids were purchased from Ikapharm (Israel), amino acids and catecholamines from Calbiochem. Reagents were bis(trimethy1silyl)acetamide (BSA), bis(trimethylsily1trifluoro)acetamide (BSTFA), trimethylchlorosilane (TMCS), 0-methoxylamine hydrochloride, perfluorobenzaldehyde, heptafluorobutyrate-imidazole, and trifluoroacetic anhydride all purchased from Pierce Chemical. Derivative formation processes were the following: corticosteroids and their metabolites were analyzed as their trimethylsilyl ethers (TMS) prepared with BSA-TMCS (1O:l) and their 0methyloxime trimethylsilyl ethers (MO-TMS) (20, 21); catalyzing conditions and a 60 "C reaction temperature were used in order to obtain a complete silylation of all hydroxyl groups present in these steroids. Aldosterone-MO-TMS derivative was made according to (22) Estrogens were as their T M S ethers prepared with BSTFA-pyridine (1:1) or as their heptafluorobutyrates
(HFB) according to (23). Catecholamine derivatives were perfluorobenzylimine-trimethylsilyl ether (PFB-TMS) (24); the modified micromethod of preparation on nanogram samples will be described elsewhere. The trifluoroacetamide butyl esters were used for the amino acids according to Gehrke et al. (25). Perdeuterated T M S ethers and MO-dg-TMS derivatives of steroids were prepared by using dl8-BSA (Merck, Sharpe and Dohme, Montreal, Canada) (26); in some instances dg-TMCS has been added as a catalyst but usually the excess of MO hydrochloride catalyzed the reaction at 60 "C and persilylation was reached after a few hours. Kinetics of exchange of TMS groups between deuterated T M S derivatives and protonated T M S reagent (BSA) has been followed by using the total mass spectrum of the d-labeled TMS steroid or for accurate measurements by means of MID. Preparation of Biological Samples. Preparation of steroid extracts for M F measurements has been made essentially by conventional methods (see for instance, ref. 27 and 28). Blood plasma was submitted to delipidation by methanol a t -18 "C for 24 hr (28). This method was also used for adrenals after homogenization in methanol-water (70:30). In both cases, the steroids were extracted by methylene chloride (two times) and ethyl acetate. The recovery of steroids from blood and plasma was evaluated by using 1%-corticosterone; total yield was around 85%. Estrogens were extracted from delipidated blood by ether and purified by partition between the alkaline water phase and the benzene hexane phase. The aqueous phase was again extracted by ether. The mean recovery of estradiol was 70%. Free and conjugated steroids were extracted from liver homogenate by chloroform-methanol (1: 1). The extract after delipidation was submitted to enzymatic hydrolysis (p-glucoronidase and sulfatase from Helix Pomatia), and the free steroids were extracted by methylene chloride and ethyl acetate. Derivatives for gas phase analysis were performed according to the forementioned method. Silanized glassware has been used. For the extraction of catecholamines from the adrenals, the method using essentially the adsorption properties of alumina toward the catecholamines (29) has been employed. The preparation of methylated amino acids from heart cell culture was the following: seven Petri dishes of heart-beating cells have been used; each 75 cm2 Petri dish contained initially 4.106 cells washed on the plates with NaCl 0.8% solution, then they were scraped with a rubber policeman and suspended in 10-15 ml of a pH 6.8, 0.15M phosphate, 0.3M KCI buffer. Cells were disrupted by freezing. The homogenate was centrifuged at 70,000 g for 1 hr. The supernatant was diluted tenfold with distilled water to allow precipitation of myosin which was recovered by centrifugation. The precipitate was hydrolyzed by 6 N HC1; the sample was evaporated to dryness by a nitrogen stream and submitted to derivative formation for gas phase analysis. The detailed experimental procedure is described in the dissertation thesis of one of us (30). Other experimental details are in the captions of the figures.
RESULT AND DISCUSSION General Conditions for Mass Fragmentography of Steroid Metabolites. General characteristics of mass fragmentography have been discussed in several papers (6, 7, 9); they are summarized in Table I, with special reference to multiple ion detection of steroids. Figure 1 illustrates the resolution power of MF; the sample submitted to gas phase analysis is the product of the reaction of estradiol with equal volumes of BSA and
Horning, W. J. A . Vandenheuvel, and B. G. Creech, Methods Biochem. Anal., 11, 69 (1962). (17) G . A . F. M . Rutten and J . A. Luyten, J . Chromatogr., 74, 177
Maurne, G. M . Maurne. J. Durand, and P. Padieu, J. Chromatogr., 58, 277 (1971). (24) A . C. Moffat and E. C. Horning. Biochem. Biophys. Acta, 222, 248
(1972). (18) P. M . J . Van den Berg and T. P. H . Cox, Chromatographia, 5 , 301 (1972). (19) B. F. Maurne and J . A. Luyten, J. Chromatogr. Sci., accepted for
(25) (26)
(16) E. C.
publication. (20) W. L. Gardiner and E. C . Horning, Biochem. Biophys. Acta, 115,
524 (1966). (21) J . P. Thenot and E. C. Horning, Anal. Lett., 5 , 21 (1972). (22) E. C. Horning and 8. F. Maurne, J. Chrornatogr. Sci., 7 , 411 (1969).
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(23) B. F.
(1970).
c . W . Gehrkeand D. Roach, J . Chromatogr., 4, 269 (1969).
J . A. McCloskey. R . N. Stillwell, and A. M . Lawson, Anal. Chem.,
40, 233 (1968). (27) D. Janne, R. V i h k o , J. Sjovall, and K . Sjovall, Clin. Chim. Acta, 23, 405 (1969). (28) H. Adlercreutz.Acta Med. Scand. Suppi., 412, 123 (1964). (29) K. Irnai. M . Sugiura, and 2. Tarnura. Chem. Pharm. Bull., 19, 409 (1971). (30) F. Barbier, These de 3erne Cycle Science, Dijon. 1972.
Table I . Specifications for Mass Fragrnentographic Analysis Analytical characteristics
Separativity
ESTRADIOL TMS, dQTMS
Evaluation for mass fragmentography
Depends on the efficiency of the GLC column (packed or capillary) Separation of unresolved GLC peaks
Sensitivity
Better than with conventional magnetic scanning Detection threshold = 0.1 pmol (depends on the nature of analyzed substances and derivatives
Specificity
Relative retention times or methylene unit values Response to specific fragments r n , / e , rnzle, m / e Values of their intensity rations i l / i 2 . idi3
Quantitative evaluation
Linear response to the mass Use of labeled internal standard (IS), Le., deuterated IS
Sampling frequency
SID = continuous M I D = about 3 H z per ion New MID (40):40Hz
dl8-BSA; the reaction products are eluted into the same GLC peak (left part); the mass fragmentogram (right part) shows the separation of three substances, estradioldi-ds-TMS at m / e 416 (molecular ion), estradiol-TMSdg-TMS a t m/e 425, and estradiol-di-ds-TMS at m / e 434; perdeutero-TMS derivatives are usually employed for the elucidation of fragment ion structures (26). Reproducible techniques to prepare glass capillary columns suitable for steroid analyses are now available (17, 31, 32). These capillaries are of great interest for GLCMS work on steroids (33). Their use in mass fragmentography (19) brings a simultaneous enhancement of the resolution of structurally close steroids and of the sensitivity of their detection. For example, estrone, the two estradiol isomers, and the four estriol isomers can be analyzed on a SE-30 glass-capillary column coupled to a mass spectrometer; a mixture of‘ these seven estrogens (40 ng of each) as T M S ethers is completely resolved at 210 “C and the SID trace obtained by using the fragment ion a t m / e 129 shows a far off-scale response for estradiol and oestriol peaks (19). The chromatography of the same mixture with a 6000 theoretical plate packed column coated with 1% SE-30 fails to separate the 16a,l7a-estriol and its 16a,17P isomer. Quantitation down to the nanomole and the picomole level with a high specificity can be achieved by using mass fragmentography and an internal standard (IS). One of the three fragment ions is used for the internal standard detection. Comparison of the peak area of the internal standard, of which a known amount was added, and the area of the biological peak allows its quantitation; a correction must be eventually made for the differences in fragment ion intensities. Structural analogs and 2Hor 15N labeled compounds as internal standard have been reported in drug studies (34) and deuterated prostaglandins (31) J. Grob and G. Grob, Chrornatographia. 4, 422 (1971). (32) M . Novotny, R . Segura, and A . Zlatkis, Anal. Chem., 44, 9 (1972), and references therein. (33) J. A. Vollmin. Chromatographia, 3, 238 (1970). (34) T. E. Gaffuey, C. G. Hamrnar. €3. Holmstedt, and R. E. McMahon, Anal. Chem., 43, 307 (1971).
MID
Xl
12
8 time
4
mn
f
8
time
9
rnn
Figure 1. GLC-MS analysis of reaction products of estradiol with amixtureof BSAandd18-BSA( 1 : i ) Left part, detection by TIC: right part, M I D performed simultaneously; GLC conditions, 1% SE-30 column at 210 “C. In this experiment differences in the proportions of di-dg-TMS and di-TMS peaks may be due to differences in reactivity between d18-BSA and BSA reagents
as internal standards have been described (35). We used three kinds of internal standard in this study. 1. A nonbiological substance with a different retention time and/or a different monitored fragment ion; an example will be given in our studies of catecholamines. 2. A nonbiological isomer of one of the substances submitted to the analysis. This isomer must be chromatographically separated from the biological one since they are also “isofragment” compounds. Epiestradiol and epiestriols are used for estrogen measurement. 3. The very compound(s) submitted to the analysis but suitably labeled by heavy isotopes. This internal standard is an almost ideal one since its retention behavior is the same (or almost the same) as the one of the cold substance. Especially if the biological compound shows an adsorption behavior during the GLC-MS process, losses are diluted by adding several times more of the labeled compound (35). This permits the detection of steroid amounts below the adsorption threshold which is reached when the cold molecule is analyzed alone. The stable isotope labeling must be different, with respect to mass, from the labeling of the extraction standard(s), and/or the labeled metabolic precursor(s) if they are used. Therefore, it is interesting to use a labeled derivative of the reference compound besides the cold derivative of the biological substance. For this reason we propose the use, as internal standard, of the perdeuterotrimethylsilyl dS-TMS derivative of ,hydroxysteroids. A known amount of ds-TMS derivative is added to the biological T M S steroid mixture before the injection into the GLC-MS system. Fragments a t m / e F and mle F + 9n (where n is the number of the derivated hydroxyl groups present in the choosen fragment) are recorded and the corresponding areas are directly compared (generally there is no isotopic effect in the fragmentation process) for quantitation. It is obviously necessary to check beforehand that the ion at m / e F is of negligible abundance in the mass spectrum of the labeled derivative. Furthermore, a ds-TMS group, substituting the hydrogen of a nonphenolic alcohol function, is generally not exchangeable with the protonated TMS reagent of the biological mix(35) B. Samuelsson, M. Hamberg. and C. C. Sweeley, Anal. Biochem., 38, 301 (1970).
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
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1 I
/’
- 3 M e E2
TIME
hr
Figure 2. Rate of exchange in O/OO of one ds-TMS against one protonated TMS group when (dg-TMS) derivatives are allowed to react with BSA for several reaction times E2 = estradiol, 3 ME2 = 3-rnethylestradiol, THB = 3a,llp,Pl-trihydroxy-5fi-pregnan-20-one as dg-TMS or Mo-dg-TMS derivatives
ture. Figure 2 shows the rate of exchange in
0’00
of one
(9-
TMS group against one T M S group for estradiol, 3methylestradiol, and tetrahydrocorticosterone perdeuteroT M S derivatives after different reaction times with protonated BSA a t room temperature. Except for estradiol, the exchange is negligible for a time shorter than 10 hr; detectable exchange was not observed in the injector and/or the column when the ds-TMS derivative was mixed with BSA just before the injection. For estradiol analysis, it is suitable to introduce the d-labeled standard in the sample just before its introduction in the GLC-MS system or to use the 3-methyl-l7p-TMS derivative. An important question is: how does the response of the mass spectrometer as a detector increase when the amount of the substance injected in the GLC column is increased? By using the 3-methylestradiol-TMS ether as a model compound, the plot of the intensity of the molecular ion ( m / e 358) against the introduced amount is linear from 100 pg to 100 ng; the straight line is going through zero showing that adsorption of the studied derivative in the GLC-MS system is very weak. The detection threshold for this steroid is 25 pg. Applications to Cellular and Extracellular Amino Acids, Steroids, a n d Catecholamines. I t is mainly for the evaluation of metabolites in cell cultures that we have developed micromethods of analysis of these substances. But it is also fundamental to know for the donor animal, how much of each hormone is present in the gland, how much is transported by the blood, how much is inside the receptor tissue and the target cell, and what is the secretion rate under normal or established conditions of response of the harmonogenic tissue. Excretion rate can be obtained by perfusing the organism, or the isolated organ, or a flow-culture dish with a steroid labeled by heavy isotope on the ring (or other substrate). Biological applications are given in the following text. Methylated Amino Acids in Heart Cell Culture. Some rare amino acids are specific of specialized proteins; for instance N-t-monomethyllysine and N-t-trimethyllysine are present in cardiac myosin and 3-methylhistidine in cardiac actin. As the N-t-methyllysines are chemical markers of the myosin, their qualitative analysis allows an identification of the protein and their quantitative determination is a means to approach the methylating process of the myosin and its evolution during the development of myocardium function. The GLC separation between lysine and N-t-monomethyllysine as their butyl-TFA derivatives is satisfactory 1076
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on a 1% OV-17 column. But as the concentration of N-tmonomethyllysine in cardiac myosin hydrolysate is very low, conventional GLC detectors do not allow its detection. In mass fragmentography, the lysine is detected by using the fragment ion a t m / e 320 (M - 74 = M - butyl alcohol) which represents 4% of the base peak, the N-tmonomethyllysine by using fragment ions a t m / e 334 (M - 74) (31%) and a t 307 (M - 101 = M COOH(CH2)&Hs) (17%). Our results show that there is no N-t-monomethyllysine in actin from the rat heart. But we have shown that there is one residue of N-c-monomethyllysine in one molecule of the myosin isolated from the whole heart, this corresponds to one residue of N-tmonomethyllysine for 620 residues of lysine. The injected sample for assay is as low as 4 p M of myosin, corresponding to 4 p M of N-t-monomethyllysine measured on the mass fragmentogram. Figure 3 shows the same method applied to the analysis of myosin from heart cell culture. On the left part of the figure one can see the gas chromatographic separation of some amino acids from the myosin hydrolysate; the detection is made by means of total ionization current (TIC), the arrow shows the place where N-t-monomethyllysine might be eluted. The right part of the figure is the mass fragmentogram of the same extract; the lysine peak can be measured on the less sensitive scale ( X l ) with the ion a t m / e 320 (broken line with points); the N-t-monomethyllysine peak can be quantitated on the most sensitive scale (x100) with the ion a t m / e 307 (solid line). This experiment shows that the molecular ratio of N-t-monomethyllysine/lysine is about the same as in the organ. Corticosteroid Hormones in Adrenals and Blood. GLC analysis of corticosteroid hormones which have kept the 4-en-3-one structure have not been investigated to the same extent as their metabolites such as 17-oxosteroids and reduced corticosteroids. This is mainly because of their low concentration in biological materials. We have developed a method for the separation of 13 steroids of the biogenetic pathway from pregnenolone to 17-deoxycorticosteroids, 17-hydroxycorticosteroids, and aldosterone. 0-Methyloxime (M0)-per-TMS derivatives were the best derivatives for all corticosteroids that we submitted to gas phase analysis. The three next figures show three steps in the analysis of a total steroid extract of adrenals; from the first to the third step of analysis, a striking increase in sensitivity, resolution, and specificity is obvious. Figure 4 shows gas chromatograms obtained with a combined GLC-MS instrument and recorded by using the total ionization current (TIC) detection (sensitivity is about the same as by use of a flame ionization detection); in Figure 4a we see a good separation between nine important reference corticosteroids. Figure 4b is an adrenal extract (the equivalent of two rat adrenal glands was injected); the important peak of cholesterol does not affect the resolution of small peaks of corticosteroids, but the latter peaks can hardly be quantitated because of their small area, and their homogeneity must be checked. Figure 5 shows the mass fragmentograms recorded simultaneously with chromatograms seen in Figure 4 while monitoring two fragment ions: m / e 103 characteristic of the 21-CH2-OTMS group and m / e 100 corresponding to C-16 apd C-17 of the D ring plus the side chain of progesterone type. Peaks of deoxycorticosterone (C) ( m / e 103) and llp-hydroxyprogesterone (111) ( m / e 100) unresolved on the TIC recording are resolved on the mass fragmentogram (upper panel). On the mass fragmentogram of the
CELLULAR
MYOSIN
1
GLC 1% OV17
160'
1
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I \
:
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Figure 3. Assay of N-6-monoethyllysine (CHsLys) in myosin hydrolysate from cultured heart cells. Left part: detection some amino acids. GLC conditions
by
the TIC of
1% OV-17 column (10-ft X 3-mm i.d.): column temperature 160 "C. Right part: mass fragmentogram showing the lysine peak, ion at m / e 320 (broken line with points), and its isotopic ion at m / e 321 (broken line) and the CH3Lys peak, ion at m / e = 307 (solid line). The sensitivity ratio between the two scales is 1 : 100. The voltage of the electron multiplier was 2.7 kV (gain 6 ) , The CH3Lys peak corresponds to about 1OpM
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Figure 4. Separation of corticosteroidswith a GLC-MS instrument TIC employed for recording. The column temperature was programmed from 200 " C at 1.5 "C per min. Steroids were analyzed as their MO-per-TMS derivatives. A = pregnenolone, B = progesterone, C = 11-deoxycorticosterone, D = 11-dehydrocorticosterone, E = corticosterone, I = 17cu-hydroxypregnenolone, I I = 17cu-hydroxyprogesterone, I l l = 1lp-hydroxyprogesterone, I V = 11-deoxycortisol, V = cortisol. The right part (a) shows the reference compounds and the left part (b) shows the steroids from a total extract of rat adrenals. The injection was equivalent to 2 adrenai glands. Chol = cholesterol-TMS, internal standards are IS1 = tetrahydrocorticosterone as MO-per-TMS derivative, IS2 = cholesteryl butyrate; peaks 1-14 are unknown. D' = 18-hydroxydeoxycorticosterone
adrenal extract (lower panel), the cholesterol peak is nearly undetected compared to the TIC recording; shoulders are resolved (see, for instance, progesterone (B) and peak No. 8); corticosterone (E), 11-dehydrocorticosterone (D), 18-hydroxydeoxycorticosterone (D'), deoxycorticosterone (C), progesterone (B), and pregnenolone (A) give important responses; other peaks (1-14) are not yet identified but they contain fragment(s) a t m / e 100 and/or 103 in their spectrum.
Figure 6 shows a more specific assay of corticosterone in adrenal extract. Corticosterone-di-MO-di-da-TMS was added to the sample before the injection. Biological corticosterone is monitored on the mass spectrometer by using the fragment ion at m / e 517 (M - 31). The same type of fragment is used for the d-labeled standard corticosterone but its m / e value is increased by 18 mass units because of its deuterium content; this ion a t m / e 535 is recorded on the left part of the mass fragmentogram. A 100-ng sample ANALYTICAL CHEMISTRY, VOL. 45, NO. 7 , J U N E 1973
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A N A L Y T I C A L CHEMISTRY, VOL. 45, No. 7 , JUNE
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